Tuberculosis is a leading health problem worldwide. Mycobacterium tuberculosis virulence is based on its capability to reside inside macrophages. Host defense includes reactive oxygen species, reactive nitrogen species, removal of metal micronutrients and lowered pH. The microorganism survives, in part, by secreting various metalloenzymes (SODs, catalases, etc.) to overcome macrophage defenses. At the center of its pathogenic strategy is the handling of involved heavy metals (Cu+, Zn2+, Fe2+, Mn2+, etc.), protons and perhaps alkali metals such as K+ and Mg2+. M. tuberculosis genome reveals the presence of numerous metal transporters. Among these, the more prominent are P-type ATPases. M. tuberculosis has 12 genes coding for these proteins (named Ctp, cation transport protein): 3 Cu+-ATPases, a K+-ATPase, 4 heavy metal ATPases of unknown substrate and 4 ATPases that likely transport H+ or alkali metals. We hypothesize that the pathophysiological role of these transporters is primarily determined by their substrate specificity. Metal specificities of a subset of these P-type ATPases will be determined by expressing these in E. coli, examining bacterial gain of function, and measuring the enzymatic and transport activity. Previous studies suggest that CtpC and CtpD are particularly relevant for in vivo (macrophage) M. tuberculosis growth. Testing this, M. tuberculosis mutants will be constructed along with strains overexpressing these proteins. Their virulence (in mice and cultured macrophages) and fitness to growth in various media will be assessed. The impact of mutating and overexpressing these proteins on M. tuberculosis metal homeostasis and its ability to respond to the host redox attack will be determined. Results form these studies will provide an initial appreciation of these proteins importance and enable the generation meaningful testable hypothesis on the biological function of these transporters. PUBLIC HEALTH RELEVANCE: Approximately one-third of the world's population is infected with Mycobacterium tuberculosis. In spite of an available vaccine and effective treatments, there are 2 million tuberculosis-related deaths per year. This project will characterize a group of proteins that appear essential for tuberculosis virulence.